利用重组工程技术标记鼠疫菌染色体基因

发布时间：2019-05-15 05:44

【摘要】：背景：鼠疫是由鼠疫耶尔森氏菌(简称鼠疫菌)引起的自然疫源性烈性传染病，可导致腺鼠疫和肺鼠疫。在人类历史上因鼠疫而死亡的人数达数以亿计。目前，鼠疫虽得到有效地控制，但鼠疫菌作为最为烈性的生物战剂和生物恐怖剂为恐怖组织所利用的可能性仍然存在。随着对多种微生物基因组测序工作的完成，经典的遗传学研究方式也在发生变化。基于重组工程技术的一步法突变和标记染色体基因已成为研究基因功能的重要手段。为加快研究鼠疫菌染色体基因及其产物的功能，我们将重组工程技术应用于标记鼠疫菌染色体基因。方法：将标签序列合成于引物中，通过融合PCR的方法将标签序列与卡那霉素或氯霉素抗性筛选基因连接构建融合模块，再将标签和抗性基因的融合模块克隆到pBluescript载体，获得模板载体。根据染色体目的基因的末端序列及其下游序列设计带短同源臂引物(40-50bp)，从模板载体上PCR扩增染色体目的基因的重组标签盒。将标签盒电转化进入表达Red重组系统的鼠疫菌感受态细胞，经同源重组表位标签融合到目的基因的下游，由此产生的C末端标记的融合蛋白可经标准的免疫学方法检测。在本研究中，我们采用的表位标签是Myc和6×His组和标签，称为MH标签，所采用的抗性筛选标记是卡那霉素基因，标记的是鼠疫菌染色体rpoS基因(sigam因子)。重组标记克隆进行DNA序列分析，C末端标记的融合蛋白的表达通过针对His标签的单克隆抗体进行Western blot验证。结果：Myc-His组合标签和卡那霉素抗性筛选基因的PCR融合产物成功的克隆到pBluescript载体，获得模板载体pbluescript-MH。以该质粒作为模板，扩增标记标签盒，，对鼠疫菌的rpoS基因进行了标记，利用针对组氨酸标签的抗体能够检测到rpoS基因的表达。结论：基于重组工程的基因标记技术易操作，检测目的蛋白表达灵敏度高，利用标签的特性可以为鼠疫菌及其他细菌基因功能研究提供一个有价值的研究手段。
[Abstract]:Background: plague is a natural epidemic severe infectious disease caused by Yersinia pestis (Yersinia pestis), which can lead to gland plague and pulmonary plague. Hundreds of millions of people have died from plague in human history. At present, although plague is effectively controlled, the possibility that plague bacteria can be used by terrorist organizations as the most powerful biological warfare agent and bioterrorism agent still exists. With the completion of genome sequencing of a variety of microorganisms, the classical genetic research methods are also changing. One-step mutation and marker chromosome gene based on recombinant engineering technology has become an important means to study gene function. In order to speed up the study of the function of Yersinia pestis chromosome gene and its products, we applied the recombinant engineering technique to label the chromosome gene of Yersinia pestis. Methods: the tag sequence was synthesized into primers, and the tag sequence was ligated with kanamycin or chloramphenicol resistance screening gene by fusion PCR to construct the fusion module, and then the fusion module of label and resistance gene was cloned into pBluescript vector. The template carrier is obtained. According to the terminal sequence of chromosome target gene and its downstream sequence, short homologue primers (40-50bp) were designed to amplify the recombinant label box of chromosome target gene from template vector by PCR. The label box was electrotransformed into Yersinia pestis cells expressing Red recombinant system and fused to the downstream of the target gene by homologous recombinant epitope tag. The resulting C-terminal labeled fusion protein could be detected by standard immunological method. In this study, the epitope tags used were Myc and 6 脳 His groups and tags, called MH tags. The resistance screening markers used were kanamycin gene and Yersinia pestis chromosome rpoS gene (sigam factor). The expression of C-terminal labeled fusion protein was confirmed by Western blot with monoclonal antibody against His tag. Results: the PCR fusion product of Myc-His combination tag and kanamycin resistance screening gene was successfully cloned into pBluescript vector, and the template vector pbluescript-MH. was obtained. Using the plasmid as template, the tag box was amplified to mark the rpoS gene of Yersinia pestis. The expression of rpoS gene could be detected by using the antibody against histidine label. Conclusion: the gene labeling technique based on recombinant engineering is easy to operate and has high sensitivity to detect the expression of the target protein. The characteristics of the tag can provide a valuable research method for the study of gene function of Yersinia pestis and other bacteria.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

【共引文献】